Aurora kinase inhibitors for inhibiting mitotic progression

ABSTRACT

The present invention relates to compounds and methods for the treatment of cancer. In particular, the invention provides potent inhibitors of Aurora A kinase, pharmaceutical compositions comprising the compounds, and methods of using the compounds for the treatment of cancer.

PRIORITY CLAIM

The present application is a continuation of U.S. patent applicationSer. No. 13/217,729, filed Aug. 25, 2011, now issued as U.S. Pat. No.9,988,384, which is a division of U.S. patent application Ser. No.11/985,277, filed Nov. 14, 2007 , which claims the benefit of U.S.Provisional Pat. Application Ser. No. 60/859,340, filed Nov. 16, 2006(abandoned). The entire contents of each of the above-referenced patentapplications are incorporated herein by this reference.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), the present specification makesreference to a Sequence Listing submitted electronically in the form ofa text file (entitled “2003886-0958 SL.txt,” created on Oct. 18, 2018,962 bytes in size). The entire contents of the Sequence Listing areherein incorporated by reference, with the intention that, uponpublication (including issuance), this incorporated sequence listingwill be inserted in the published document immediately before theclaims.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to compounds and methods for the treatment ofcancer. In particular, the invention provides a compound that inhibitsAurora kinase enzymes, pharmaceutical compositions comprising thecompound, and methods of using the compound for the treatment of cancer.

Background of the Invention

According to the American Cancer Society, an estimated 1.4 millionAmericans were newly-diagnosed with cancer in 2004 and about 560,000victims died from the disease. While medical advance have improvedcancer survival rates, there is a continuing need for new and moreeffective treatment.

Cancer is characterized by uncontrolled cell reproduction. Mitosis is astage in the cell cycle during which a series of complex events ensurethe fidelity of chromosome separation into two daughter cells. Severalcurrent cancer therapies, including the taxanes and vinca alkaloids, actto inhibit the mitotic machinery. Mitotic progression is largelyregulated by proteolysis and by phosphorylation events that are mediatedby mitotic kinases. Aurora kinase family members (e.g., Aurora A, AuroraB, Aurora C) regulate mitotic progression through modulation ofcentrosome separation, spindle dynamics, spindle assembly checkpoint,chromosome alignment, and cytokinesis (Dutertre et al., Oncogene, 21:6175 (2002); Berdnik et al., Curr. Biol., 12: 640 (2002)).Overexpression and/or amplification of Aurora kinases have been linkedto oncogenesis in several tumor types including those of colon andbreast (Warner et al., Mol. Cancer Ther., 2: 589 (2003); Bischoff etal., EMBO, 17: 3062 (1998); Sen et al., Cancer Res., 94: 1320 (2002)).Moreover, Aurora kinase inhibition in tumor cells results in mitoticarrest and apoptosis, suggesting that these kinases are importanttargets for cancer therapy (Ditchfield, J. Cell Biol., 161: 267 (2003);Harrington et al., Nature Med., 1 (2004)). Given the central role ofmitosis in the progression of virtually all malignancies, inhibitors ofthe Aurora kinases are expected to have application across a broad rangeof human tumors. There is thus a need for new Aurora kinase inhibitors.

DESCRIPTION OF THE INVENTION

Claiborne et al., International Patent Publication WO 05/111039,discloses pyrimidobenzazepine compounds with Aurora kinase inhibitoryactivity. The present inventors have now discovered pyrimidobenzazepinecompounds with unexpectedly superior potency against Aurora A kinase.The claimed compounds are useful for inhibiting Aurora A kinase activityin vitro and in vivo, and are especially useful for the treatment ofvarious cell proliferative diseases.

In one aspect, therefore, the invention provides a compound representedby formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R^(a) is selected from the group consisting of C₁₋₃ aliphatic,        C₁₋₃ fluoroaliphatic, -T-R¹, —R², and -T-R²;    -   T is a C1-3 alkylene chain optionally substituted with fluoro;    -   R¹ is an optionally substituted aryl, heteroaryl, or        heterocyclyl group;    -   R² is selected from the group consisting of halo, —C≡C—R³,        —CH═CH—R³, —N(R⁴)₂, and —OR⁵;    -   R³ is hydrogen or an optionally substituted aliphatic, aryl,        heteroaryl, or heterocyclyl group;    -   each R⁴ independently is hydrogen or an optionally substituted        aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R⁴ on        the same nitrogen atom, taken together with the nitrogen atom        form an optionally substituted 5- to 6-membered heteroaryl or 4-        to 8-membered heterocyclyl ring having, in addition to the        nitrogen atom, 0-2 ring heteroatoms selected from N, O, and S;    -   R⁵ is hydrogen or an optionally substituted aliphatic, aryl,        heteroaryl, or heterocyclyl group; and    -   R^(b) is selected from the group consisting of fluoro, chloro,        —CH₃, —CF₃, —OH, —OCH₃, —OCF₃, —OCH₂CH₃, and —OCH₂CF₃.

In some embodiments, R¹ is a 5- or 6-membered aryl, heteroaryl, orheterocyclyl ring optionally substituted with one or two substituentsindependently selected from the group consisting of halo, C₁₋₃aliphatic, and C₁₋₃ fluoroaliphatic. In certain embodiments, R¹ is aphenyl, furyl, pyrrolidinyl, or thienyl ring optionally substituted withone or two substituents independently selected from the group consistingof halo, C₁₋₃ aliphatic, and C₁₋₃ fluoroaliphatic.

In some embodiments, R³ is hydrogen, C₁₋₃ aliphatic, C₁₋₃fluoroaliphatic, or —CH₂—OCH₃.

In some embodiments, R⁵ is hydrogen, C₁₋₃ aliphatic, or C₁₋₃fluoroaliphatic.

In certain embodiments, Ra is halo, C₁₋₃ aliphatic, C₁₋₃fluoroaliphatic, —OH, —O(C₁₋₃ aliphatic), —O(C₁₋₃ fluoroaliphatic),—CH═CH—R³, or an optionally substituted pyrrolidinyl, thienyl, furyl, orphenyl ring, wherein R³ is hydrogen, C₁₋₃ aliphatic, C₁₋₃fluoroaliphatic, or —CH₂—OCH₃. In certain particular embodiments, R^(a)is selected from the group consisting of chloro, fluoro, C₁₋₃ aliphatic,C₁₋₃ fluoroaliphatic, —OCH₃, —OCF₃, —C═C—CH₃, —CH═CH₂, —CH═CHCH₃,N-methylpyrrolidinyl, thienyl, methylthienyl, furyl, methylfuryl,phenyl, fluorophenyl, and tolyl.

Table 1 shows specific examples of compounds of formula (I).

TABLE 1 Aurora Kinase Inhibitors

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

The compounds in Table 1 above also may be identified by the followingchemical names:

Chemical Name 14-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 24-{[9-ethynyl-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 34-({9-chloro-7-[2-fluoro-6-(trifluoromethoxy)phenyl]-5H-pyrimido[5,4-d][2]benzazepin-2-yl}amino)-2-methoxybenzoic acid 44{[7-(2-fluoro-6-methoxyphenyl)-9-(1-methyl-1H-pyrrol-2-yl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 54-{[7-(2-fluoro-6-methoxyphenyl)-9-(4-methyl-3-thienyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 64-{[7-(2-fluoro-6-methoxyphenyl)-9-(3-methyl-2-furyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 74-({9-ethynyl-7-[2-fluoro-6-(2,2,2-trifluoroethoxy)phenyl]-5H-pyrimido[5,4-d][2]benzazepin-2-yl}amino)-2-methoxybenzoic acid 84-{[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 94-{[7-(2-fluoro-6-methoxyphenyl)-9-(2-methylphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 104-{[7-(2-fluoro-6-methoxyphenyl)-9-prop-1-yn-1-yl-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 114-{[7-(2-fluoro-6-methoxyphenyl)-9-vinyl-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 124-{[7-(2-fluoro-6-methoxyphenyl)-9-(2-fluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 134-{[7-(2-fluoro-6-methoxyphenyl)-9-(3-methoxyprop-1-yn-1-yl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 144-({7-(2-fluoro-6-methoxyphenyl)-9-[(1E)-prop-1-en-1-yl]-5H-pyrimido[5,4-d][2]benzazepin-2-yl}amino)-2-methoxybenzoic acid 154-({9-chloro-7-[2-fluoro-6-(2,2,2-trifluoroethoxy)phenyl]-5H-pyrimido[5,4-d][2]benzazepin-2-yl}amino)-2-methoxybenzoic acid 164-{[7-(2-fluoro-6-methoxyphenyl)-9-(2-furyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 174-{[9-chloro-7-(2-fluoro-6-hydroxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid 184-{[7-(2-fluoro-6-methoxyphenyl)-9-phenyl-5H-pyrimido[5,4-d][2]benzazepin-2- yl]amino}-2-methoxybenzoic acid

In one embodiment, the compound of formula (I) is4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoicacid or a pharmaceutically acceptable salt thereof. In a particularembodiment, the compound of formula (I) is sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoate.

Unless otherwise stated, structures depicted herein are meant to includecompounds which differ only in the presence of one or more isotopicallyenriched atoms. For example, compounds having the present structureexcept for the replacement of a hydrogen atom by a deuterium or tritium,or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of the invention.

The term “aliphatic” or “aliphatic group”, as used herein, means asubstituted or unsubstituted straight-chain, branched or cyclic C₁₋₁₂hydrocarbon, which is completely saturated or which contains one or moreunits of unsaturation, but which is not aromatic. For example, suitablealiphatic groups include substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl, alkynyl groups and hybrids thereof, such as(cylcoalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “cycloaliphatic”, used alone or as part of a larger moiety,refers to a saturated or partially unsaturated cyclic aliphatic ringsystem having from 3 to about 14 members, wherein the aliphatic ringsystem is optionally substituted. In some embodiments, thecycloaliphatic is a monocyclic hydrocarbon having 3-8 or 3-6 ring carbonatoms. Nonlimiting examples include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In someembodiments, the cycloaliphatic is a bridged or fused bicyclichydrocarbon having 6-12, 6-10, or 6-8 ring carbon atoms, wherein anyindividual ring in the bicyclic ring system has 3-8 members.

In some embodiments, two adjacent substituents on the cycloaliphaticring, taken together with the intervening ring atoms, form an optionallysubstituted fused 5- to 6-membered aromatic or 3- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the term “cycloaliphatic” includesaliphatic rings that are fused to one or more aryl, heteroaryl, orheterocyclyl rings. Nonlimiting examples include indanyl,5,6,7,8-tetrahydroquinoxalinyl, decahydronaphthyl, ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring. The term “cycloaliphatic” may be used interchangeablywith the terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or“carbocyclic”.

The terms “aryl” and “ar-”, used alone or as part of a larger moiety,e.g., “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refer to a C₆ to C₁₄aromatic hydrocarbon, comprising one to three rings, each of which isoptionally substituted. Preferably, the aryl group is a C₆₋₁₀ arylgroup. Aryl groups include, without limitation, phenyl, naphthyl, andanthracenyl. In some embodiments, two adjacent substituents on the arylring, taken together with the intervening ring atoms, form an optionallysubstituted fused 5- to 6-membered aromatic or 4- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the term “aryl”, as used herein,includes groups in which an aromatic ring is fused to one or moreheteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical orpoint of attachment is on the aromatic ring. Nonlimiting examples ofsuch fused ring systems include indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, fluorenyl,indanyl, phenanthridinyl, tetrahydronaphthyl, indolinyl, phenoxazinyl,benzodioxanyl, and benzodioxolyl. An aryl group may be mono-, bi-, tri-,or polycyclic, preferably mono-, bi-, or tricyclic, more preferablymono- or bicyclic. The term “aryl” may be used interchangeably with theterms “aryl group”, “aryl moiety”, and “aryl ring”.

An “aralkyl” or “arylalkyl” group comprises an aryl group covalentlyattached to an alkyl group, either of which independently is optionallysubstituted. Preferably, the aralkyl group is C₆₋₁₀ aryliC₁₋₆)alkyl,C₆₋₁₀ aryl(C₁₋₄)alkyl, or C₆₋₁₀ aryl(C₁₋₃)alkyl, including, withoutlimitation, benzyl, phenethyl, and naphthylmethyl.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., heteroaralkyl, or “heteroaralkoxy”, refer to groupshaving 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to four heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. In some embodiments, twoadjacent substituents on the heteroaryl, taken together with theintervening ring atoms, form an optionally substituted fused 5- to6-membered aromatic or 4- to 8-membered non-aromatic ring having 0-3ring heteroatoms selected from the group consisting of O, N, and S.Thus, the terms “heteroaryl” and “heteroar-”, as used herein, alsoinclude groups in which a heteroaromatic ring is fused to one or morearyl, cycloaliphatic, or heterocyclyl rings, where the radical or pointof attachment is on the heteroaromatic ring. Nonlimiting examplesinclude indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono-,bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, morepreferably mono- or bicyclic. The term “heteroaryl” may be usedinterchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or“heteroaromatic”, any of which terms include rings that are optionallysubstituted. The term “heteroaralkyl” refers to an alkyl groupsubstituted by a heteroaryl, wherein the alkyl and heteroaryl portionsindependently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclicradical”, and “heterocyclic ring” are used interchangeably and refer toa stable 3- to 7-membered monocyclic, or to a fused 7- to 10-membered orbridged 6- to 10-membered bicyclic heterocyclic moiety that is eithersaturated or partially unsaturated, and having, in addition to carbonatoms, one or more, preferably one to four, heteroatoms, as definedabove. When used in reference to a ring atom of a heterocycle, the term“nitrogen” includes a substituted nitrogen. As an example, in aheterocyclyl ring having 1-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl) or ⁺NR (as in N-substituted pyrrolidinyl). Aheterocyclic ring can be attached to its pendant group at any heteroatomor carbon atom that results in a stable structure, and any of the ringatoms can be optionally substituted. Examples of such saturated orpartially unsaturated heterocyclic radicals include, without limitation,tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.

In some embodiments, two adjacent substituents on a heterocyclic ring,taken together with the intervening ring atoms, for an optionallysubstituted fused 5- to 6-membered aromatic or 3- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the terms “heterocycle”,“heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclicmoiety”, and “heterocyclic radical”, are used interchangeably herein,and include groups in which a heterocyclyl ring is fused to one or morearyl, heteroaryl, or cycloaliphatic rings, such as indolinyl,3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, wherethe radical or point of attachment is on the heterocyclyl ring. Aheterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferablymono-, bi-, or tricyclic, more preferably mono- or bicyclic. The term“heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond between ring atoms. Theterm “partially unsaturated” is intended to encompass rings havingmultiple sites of unsaturation, but is not intended to include aryl orheteroaryl moieties, as herein defined.

The terms “haloaliphatic”, “haloalkyl”, “haloalkenyl” and “haloalkoxy”refer to an aliphatic, alkyl, alkenyl or alkoxy group, as the case maybe, which is substituted with one or more halogen atoms. As used herein,the term “halogen” or “halo” means F, Cl, Br, or I. The term“fluoroaliphatic” refers to a haloaliphatic wherein the halogen isfluoro.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms is replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group. An alkylene chain also may be substitutedat one or more positions with an aliphatic group or a substitutedaliphatic group.

The term “substituted”, as used herein, means that a hydrogen radical ofthe designated moiety is replaced with the radical of a specifiedsubstituent, provided that the substitution results in a stable orchemically feasible compound. The phrase “one or more substituents”, asused herein, refers to a number of substituents that equals from one tothe maximum number of substituents possible based on the number ofavailable bonding sites, provided that the above conditions of stabilityand chemical feasibility are met. Unless otherwise indicated, anoptionally substituted group may have a substituent at eachsubstitutable position of the group, and the substituents may be eitherthe same or different.

An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyland the like) or heteroaryl (including the heteroaryl moiety inheteroaralkyl and heteroaralkoxy and the like) group may contain one ormore substituents. Examples of suitable substituents on the unsaturatedcarbon atom of an aryl or heteroaryl group include —halo, —NO₂, —CN,—R*, —C(R*)═C(R*)₂, —OR*, —SR^(o), —S(O)R^(o), —SO₂R^(o), —SO₃R^(o),—SO₂N(R⁺)₂, —N(R⁺)₂, —NR+C(O)R*, —NR+C(O)N(R⁺)₂, —NR+CO₂R^(o), —O—CO₂R*,—OC(O)N(R⁺)₂, —O—C(O)R*, —CO₂R*, —C(O)—C(O)R*, —C(O)R*, —C(O)N(R⁺)₂,—C(O)N(R⁺)C(═NR⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—N(R⁺)—C(O)R*, —C(═NR⁺)—N(R⁺)₂,—C(═NR⁺)—OR*, —N(R⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—N(R⁺)₂, —NR⁺SO₂R^(o),—NR⁺SO₂N(R⁺)₂, —P(O)(R*)₂, —P(O)(OR*)₂, —O—P(O)—OR*, and—P(O)(NR⁺)—N(R⁺)₂; or two adjacent substituents, taken together withtheir intervening atoms, form a 5-6 membered unsaturated or partiallyunsaturated ring having 0-3 ring atoms selected from the groupconsisting of N, O, and S.

An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyland the like) or heteroaryl (including the heteroaryl moiety inheteroaralkyl and heteroaralkoxy and the like) group may contain one ormore substituents. Examples of suitable substituents on the unsaturatedcarbon atom of an aryl or heteroaryl group include -halo, —NO₂, —CN,—R*, —C(R*)═C(R*)₂, —C═C—R*, —OR*, —SR^(o), —S(O)R^(o), —SO₂R^(o),—SO₃R^(o), —SO₂N(R⁺)₂, —N(R⁺)₂, —NR⁺C(O)R*, —NR⁺C(O)N(R⁺)₂,—NR+CO₂R^(o), —O—CO₂R*, —OC(O)N(R⁺)₂, —O—C(O)R*, —CO₂R*, —C(O)—C(O)R*,—C(O)R*, —C(O)N(R⁺)₂, —C(O)N(R⁺)C(═NR⁺)—N(R⁺)₂,—N(R⁺)C(═NR⁺)—N(R⁺)—C(O)R*, —C(═NR⁺)—N(R⁺)₂, —C(═NR⁺)—OR*,—N(R⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—N(R⁺)₂, —NR⁺SO₂R^(o), —NR+SO₂N(R⁺)₂,—P(O)(R*)₂, —P(O)(OR*)₂, —O—P(O)—OR*, and —P(O)(NR⁺)—N(R⁺)₂; or twoadjacent substituents, taken together with their intervening atoms, forma 5-6 membered unsaturated or partially unsaturated ring having 0-3 ringatoms selected from the group consisting of N, O, and S.

Each R⁺, independently, is hydrogen or an optionally substitutedaliphatic, aryl, heteroaryl, or heterocyclyl group, or two R⁺ on thesame nitrogen atom, taken together with the nitrogen atom, form a 5-8membered aromatic or non-aromatic ring having, in addition to thenitrogen atom, 0-2 ring heteroatoms selected from N, O, and S. Each R*independently is hydrogen or an optionally substituted aliphatic, aryl,heteroaryl, or heterocyclyl group. Each R^(o) is an optionallysubstituted aliphatic or aryl group.

An aliphatic group or a non-aromatic heterocyclic ring may besubstituted with one or more substituents. Examples of suitablesubstituents on the saturated carbon of an aliphatic group or of anon-aromatic heterocyclic ring include, without limitation, those listedabove for the unsaturated carbon of an aryl or heteroaryl group and thefollowing: ═O, ═S, ═C(R*)₂, ═N—N(R*)₂, ═N—OR*, ═N—NHC(O)R*,═N—NHCO₂R^(o), ═N—NHSO₂R^(o), or ═N—R*, where each R* and R^(o) is asdefined above.

Suitable substituents on the nitrogen atom of a non-aromaticheterocyclic ring include —R*, —N(R*)₂, —C(O)R*, —CO₂R*, —C(O)—C(O)R*—C(O)CH₂C(O)R*, —SO₂R*, —SO₂N(R*)₂, —C(═S)N(R*)₂, —C(═NH)—N(R*)₂, and—NR*SO₂R*; wherein each R* is as defined above.

Compounds of formula (I) are inhibitors of Aurora kinase. The compoundscan be assayed in vitro or in vivo for their ability to bind to and/orinhibit an Aurora kinase. In vitro assays include assays to determineinhibition of the ability of an Aurora kinase to phosphorylate asubstrate protein or peptide. Alternate in vitro assays quantitate theability of the compound to bind to an Aurora kinase. Inhibitor bindingmay be measured by radiolabelling the inhibitor prior to binding,isolating the inhibitor/Aurora kinase complex and determining the amountof radiolabel bound. Alternatively, inhibitor binding may be determinedby running a competition experiment in which new inhibitors areincubated with Aurora kinase bound to a known radioligand. The compoundsof the invention also can be assayed for its ability to affect cellularor physiological functions mediated by Aurora kinase activity. Assaysfor each of these activities are described in the Examples and/or areknown in the art.

In another aspect, therefore, the invention provides a method forinhibiting Aurora kinase activity in a cell, comprising contacting acell in which inhibition of Aurora kinase is desired with the Aurorakinase inhibitor of formula (I) or a pharmaceutically acceptable saltthereof.

Preferably, the method according to this aspect of the invention causesan inhibition of cell proliferation of the contacted cells. The phrase“inhibiting cell proliferation” is used to denote an ability of aninhibitor of Aurora kinase to inhibit cell number or cell growth incontacted cells as compared to cells not contacted with the inhibitor.An assessment of cell proliferation can be made by counting cells usinga cell counter or by an assay of cell viability, e.g., a BrdU, MTT, XTT,or WST assay. Where the cells are in a solid growth (e.g., a solid tumoror organ), such an assessment of cell proliferation can be made bymeasuring the growth, e.g., with calipers, and comparing the size of thegrowth of contacted cells with non-contacted cells.

Preferably, the growth of cells contacted with the inhibitor is retardedby at least about 50% as compared to growth of non-contacted cells. Invarious embodiments, cell proliferation of contacted cells is inhibitedby at least about 75%, at least about 90%, or at least about 95% ascompared to non-contacted cells. In some embodiments, the phrase“inhibiting cell proliferation” includes a reduction in the number ofcontacted cells, as compare to non-contacted cells. Thus, an inhibitorof Aurora kinase that inhibits cell proliferation in a contacted cellmay induce the contacted cell to undergo growth retardation, to undergogrowth arrest, to undergo programmed cell death (i.e., apoptosis), or toundergo necrotic cell death.

The present inventors have discovered that compounds of formula (I),which are characterized by a methoxy substituent at the position orthoto the carboxylic acid substituent in Ring C and a non-hydrogensubstituent Rb in Ring B, exhibit surprising potency in cell-basedassays when compared to structurally similar compounds.

For example, Table 2 shows a comparison of compound 1 to compounds i,ii, and iii disclosed in Claiborne et al., International PatentPublication WO 05/111039. Compounds 1 and i-iii were tested in threecellular assays: (1) pT288 Aurora A autophosphorylation assay; (2) BrdUcell proliferation assay in HCT116 cells; and (3) BrdU cellproliferation assay in SW480 cells. Protocols for these assays are knownin the art, and are described in Example 6. Compounds i and ii exhibitedvery similar potency in all three assays, suggesting that addition of amethoxy substituent at the position ortho to the carboxylic acidsubstituent in Ring C has little to no effect on cellular potency. Bycontrast, compound iii exhibited significantly enhanced potency in allthree assays when compared to compound ii, suggesting that an additionalsubstituent on Ring B improves potency. In view of these data, the factthat compound 1 is more potent than compounds i and ii was notunexpected. Surprisingly, however, compound 1 also exhibits a remarkable2- to 4-fold enhancement in potency compared to compound iii. As thesedata indicate, the combination of a methoxy substituent at the positionortho to the carboxylic acid substituent and a non-hydrogen substituentRb in Ring B provides an unexpected enhancement in potency.

TABLE 2 Cellular Potency of Aurora Kinase Inhibitors BrdU BrdU pT288HCT116 SW480 Compound Structure IC₅₀ (μM) LD₅₀ (μM) LD₅₀ (μM) 1

0.005 0.03 0.41 i

0.18 0.707 6.502 ii

0.15 0.758 7.579 iii

0.018 0.13 0.94

Compound 1 also is more potent than compound iii in vivo, asdemonstrated in a mouse HCT116 human colon carcinoma xenograft model(see Example 7). The improved in vivo potency of compounds of formula(I) is expected to result in an improved therapeutic index with respectto off-target side effects.

In another aspect, therefore, the invention provides a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

If a pharmaceutically acceptable salt of the compound of the inventionis utilized in these compositions, the salt preferably is derived froman inorganic or organic acid or base. For reviews of suitable salts,see, e.g., Berge et al, J. Pharm. Sci. 66:1-19 (1977) and Remington: TheScience and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, LippincottWilliams & Wilkins, 2000.

Nonlimiting examples of suitable acid addition salts include thefollowing: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate.

Suitable base addition salts include, without limitation, ammoniumsalts, alkali metal salts, such as sodium and potassium salts, alkalineearth metal salts, such as calcium and magnesium salts, salts withorganic bases, such as dicyclohexylamine, N-methyl-D-glucamine,t-butylamine, ethylene diamine, ethanolamine, and choline, and saltswith amino acids such as arginine, lysine, and so forth. In oneembodiment, the compound of formula (1) may be formulated as thecorresponding sodium salt.

Also, basic nitrogen-containing groups may be quaternized with suchagents as lower alkyl halides, such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates, such as dimethyl,diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides, such as benzyl and phenethyl bromides and others. Water oroil-soluble or dispersible products are thereby obtained.

The term “pharmaceutically acceptable carrier” is used herein to referto a material that is compatible with a recipient subject, preferably amammal, more preferably a human, and is suitable for delivering anactive agent to the target site without terminating the activity of theagent. The toxicity or adverse effects, if any, associated with thecarrier preferably are commensurate with a reasonable risk/benefit ratiofor the intended use of the active agent.

The terms “carrier”, “adjuvant”, or “vehicle” are used interchangeablyherein, and include any and all solvents, diluents, and other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington: The Science and Practice of Pharmacy, 20th Ed., ed.A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as disodium hydrogen phosphate, potassium hydrogenphosphate, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium hydroxide and aluminum hydroxide,glycine, sorbic acid, or potassium sorbate, partial glyceride mixturesof saturated vegetable fatty acids, water, pyrogen-free water, salts orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugarssuch as lactose, glucose, sucrose, starches such as corn starch andpotato starch, cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate, powderedtragacanth; malt, gelatin, talc, excipients such as cocoa butter andsuppository waxes, oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil, glycols such aspropylene glycol and polyethylene glycol, esters such as ethyl oleateand ethyl laurate, agar, alginic acid, isotonic saline, Ringer'ssolution, alcohols such as ethanol, isopropyl alcohol, hexadecylalcohol, and glycerol, cyclodextrins, lubricants such as sodium laurylsulfate and magnesium stearate, petroleum hydrocarbons such as mineraloil and petrolatum. Coloring agents, releasing agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the composition, according to thejudgment of the formulator.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally contain solvents,diluents, and other liquid vehicles, dispersion or suspension aids,surface active agents, pH modifiers, isotonic agents, thickening oremulsifying agents, stabilizers and preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired.

According to a preferred embodiment, the compositions of this inventionare formulated for pharmaceutical administration to a mammal, preferablya human being. Such pharmaceutical compositions of the present inventionmay be administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir. The term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intravenously, or subcutaneously. The formulationsof the invention may be designed to be short-acting, fast-releasing, orlong-acting. Still further, compounds can be administered in a localrather than systemic means, such as administration (e.g., by injection)at a tumor site.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. Compositions formulated for parenteral administration may beinjected by bolus injection or by timed push, or may be administered bycontinuous infusion.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices.of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents such as phosphates orcarbonates.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The pharmaceutical compositions of this invention are particularlyuseful in therapeutic applications relating to an Aurora kinase-mediateddisorder. As used herein, the term “Aurora kinase-mediated disorder”includes any disorder, disease or condition which is caused orcharacterized by an increase in Aurora kinase expression or activity, orwhich requires Aurora kinase activity. The term “Aurora kinase-mediateddisorder” also includes any disorder, disease or condition in whichinhibition of Aurora kinase activity is beneficial. Aurorakinase-mediated disorders include proliferative disorders. Non-limitingexamples of proliferative disorders include chronic inflammatoryproliferative disorders, e.g., psoriasis and rheumatoid arthritis;proliferative ocular disorders, e.g., diabetic retinopathy; benignproliferative disorders, e.g., hemangiomas; and cancer.

Preferably, the composition is formulated for administration to apatient having or at risk of developing or experiencing a recurrence ofan Aurora kinase-mediated disorder. The term “patient”, as used herein,means an animal, preferably a mammal, more preferably a human. Preferredpharmaceutical compositions of the invention are those formulated fororal, intravenous, or subcutaneous administration. However, any of theabove dosage forms containing a therapeutically effective amount of acompound of the invention are well within the bounds of routineexperimentation and therefore, well within the scope of the instantinvention. In some embodiments, the pharmaceutical composition of theinvention may further comprise another therapeutic agent. Preferably,such other therapeutic agent is one normally administered to patientswith the disease or condition being treated.

By “therapeutically effective amount” is meant an amount sufficient tocause a detectable decrease in Aurora kinase activity or the severity ofan Aurora kinase-mediated disorder. The amount of Aurora kinaseinhibitor needed will depend on the effectiveness of the inhibitor forthe given cell type and the length of time required to treat thedisorder. It should also be understood that a specific dosage andtreatment regimen for any particular patient will depend upon a varietyof factors, including the activity of the specific compound employed,the age, body weight, general health, sex, and diet of the patient, timeof administration, rate of excretion, drug combinations, the judgment ofthe treating physician, and the severity of the particular disease beingtreated. The amount of additional therapeutic agent present in acomposition of this invention typically will be no more than the amountthat would normally be administered in a composition comprising thattherapeutic agent as the only active agent. Preferably, the amount ofadditional therapeutic agent will range from about 50% to about 100% ofthe amount normally present in a composition comprising that agent asthe only therapeutically active agent.

Compositions of the invention may be formulated in unit dosage form forease of administration and uniformity of dosage. The expression “unitdosage form” as used herein refers to a physically discrete unit ofagent appropriate for the patient to be treated. It will be understood,however, that the total daily usage of the compounds and compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. A unit dosage form for parenteraladministration may be in ampoules or in multi-dose containers.

In another aspect, the invention provides a method for treating apatient having or at risk of developing or experiencing a recurrence ofan Aurora kinase-mediated disorder. The method comprises the step ofadministering to the patient a compound or pharmaceutical compositionaccording to the invention. The compounds and pharmaceuticalcompositions of the invention can be used to achieve a beneficialtherapeutic or prophylactic effect, for example, in a patient with aproliferative disorder, as discussed above. The compound andpharmaceutical compositions of the invention are particularly useful forthe treatment of cancer.

As used herein, the term “cancer” refers to a cellular disordercharacterized by uncontrolled or disregulated cell proliferation,decreased cellular differentiation, inappropriate ability to invadesurrounding tissue, and/ or ability to establish new growth at ectopicsites. The term “cancer” includes, but is not limited to, solid tumorsand bloodborne tumors. The term “cancer” encompasses diseases of skin,tissues, organs, bone, cartilage, blood, and vessels. The term “cancer”further encompasses primary and metastatic cancers.

Non-limiting examples of solid tumors that can be treated by the methodsof the invention include pancreatic cancer; bladder cancer; colorectalcancer; breast cancer, including metastatic breast cancer; prostatecancer, including androgen-dependent and androgen-independent prostatecancer; renal cancer, including, e.g., metastatic renal cell carcinoma;hepatocellular cancer; lung cancer; including, e.g., non-small cell lungcancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinomaof the lung; ovarian cancer, including, e.g., progressive epithelial orprimary peritoneal cancer; cervical cancer; gastric cancer; esophagealcancer; head and neck cancer, including, e.g., squamous cell carcinomaof the head and neck; melanoma; neuroendocrine cancer, includingmetastatic neuroendocrine tumors; brain tumors, including, e.g., glioma,anaplastic oligodendroglioma, adult glioblastoma multiforme, and adultanaplastic astrocytoma; bone cancer; and soft tissue sarcoma.

In some other embodiments, the cancer is a hematologic malignancy.Non-limiting examples of hematologic malignancy include acute myeloidleukemia (AML); chronic myelogenous leukemia (CML), includingaccelerated CML and CML blast phase (CML-BP); acute lymphoblasticleukemia (ALL); chronic lymphocytic leukemia (CLL); Hodgkin's disease(HD); non-Hodgkin's lymphoma (NHL), including follicular lymphoma andmantle cell lymphoma; B-cell lymphoma; T-cell lymphoma; multiple myeloma(MM); Waldenstrom's macroglobulinemia; myelodysplastic syndromes (MDS),including refractory anemia (RA), refractory anemia with ringedsiderblasts (RARS), (refractory anemia with excess blasts (RAEB), andRAEB in transformation (RAEB-T); and myeloproliferative syndromes.

In some embodiments, the compound or composition of the invention isused to treat a cancer in which the activity of an Aurora kinase isamplified. In some embodiments, the compound or composition of theinvention is used to treat a patient having or at risk of developing orexperiencing a recurrence in a cancer selected from the group consistingof colorectal cancer, ovarian cancer, breast cancer, gastric cancer,prostate cancer, and pancreatic cancer. In certain embodiments, thecancer is selected from the group consisting of breast cancer,colorectal cancer, and pancreatic cancer.

In some embodiments, the Aurora kinase inhibitor of the invention isadministered in conjunction with another therapeutic agent. The othertherapeutic agent may also inhibit Aurora kinase or may operate by adifferent mechanism. In some embodiments, the other therapeutic agent isone that is normally administered to patients with the disease orcondition being treated. The Aurora kinase inhibitor of the inventionmay be administered with the other therapeutic agent in a single dosageform or as a separate dosage form. When administered as a separatedosage form, the other therapeutic agent may be administered prior to,at the same time as, or following administration of the Aurora kinaseinhibitor of the invention.

In some embodiments, the Aurora kinase inhibitor of the invention isadministered in conjunction with a therapeutic agent selected from thegroup consisting of cytotoxic agents, radiotherapy, and immunotherapy.Non-limiting examples of cytotoxic agents suitable for use incombination with the Aurora kinase inhibitors of the invention include:antimetabolites, including, e.g., capecitibine, gemcitabine,5-fluorouracil or 5-fluorouracil/leucovorin, fludarabine, cytarabine,mercaptopurine, thioguanine, pentostatin, and methotrexate;topoisomerase inhibitors, including, e.g., etoposide, teniposide,camptothecin, topotecan, irinotecan, doxorubicin, and daunorubicin;vinca alkaloids, including, e.g., vincristine and vinblastin; taxanes,including, e.g., paclitaxel and docetaxel; platinum agents, including,e.g., cisplatin, carboplatin, and oxaliplatin; antibiotics, including,e.g., actinomycin D, bleomycin, mitomycin C, adriamycin, daunorubicin,idarubicin, doxorubicin and pegylated liposomal doxorubicin; alkylatingagents such as melphalan, chlorambucil, busulfan, thiotepa, ifosfamide,carmustine, lomustine, semustine, streptozocin, decarbazine, andcyclophosphamide; thalidomide and related analogs, including, e.g.,CC-5013 and CC-4047; protein tyrosine kinase inhibitors, including,e.g., imatinib mesylate and gefitinib; antibodies, including, e.g.,trastuzumab, rituximab, cetuximab, and bevacizumab; mitoxantrone;dexamethasone; prednisone; and temozolomide.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examplesillustrate how to make or test specific compounds, and are not to beconstrued as limiting the scope of the invention in any way.

EXAMPLES

Definitions

-   AcOH acetic acid-   ATP adenosine triphosphate-   BrdU 5-bromo-2′-deoxyuridine-   BSA bovine serum albumin-   DCM dichloromethane-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   EDTA ethylenediaminetetraacetic acid-   EtOH ethanol-   HPbCD hydroxypropyl beta-cyclodextrin-   MeOH methanol-   MTT methylthiazoletetrazolium-   WST    (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene    disulfonate sodium salt)-   PKA cAMP-dependent protein kinase-   THF tetrahydrofuran-   h hours-   min minutes-   m/z mass to charge-   MS mass spectrum-   HRMS high resolution mass spectrum

Melting points were determined on a MEL-TEMP II capillary melting pointapparatus and are uncorrected. ¹H NMR spectra were recorded on a BrukerAvance 400 spectrometer. Mass spectra were obtained on a Waters ZQ 2000(3.5 kV capillary, 30 V cone) spectrometer. Elemental analysis wasperformed by Atlantic Microlab.

Example 1 Preparation of4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoicacid (1)

8-Chloro-4-[(dimethylamino)methylene]-1-(2-fluoro-6-methoxyphenyl)-3,4-dihydro-5H-2-benzazepin-5-one(iv) can be prepared as described in Claiborne et al., U.S. PatentPublication 2005-256102. 4-{[amino(imino)methyl]amino}-2-methoxybenzoicacid.HCl (v) can be prepared in a manner similar to that described inSugiki et al., International Patent Publication WO 01/042199.

Methanol (50.0 mL) was added to iv (2.39 g, 6.42 mmol), v (1.77 g, 7.21mmol), and potassium carbonate.1.5[H₂O] (2.65 g, 16.0 mmol) in a 100-mLround-bottomed flask equipped with a stirbar and a reflux condenser. Thereaction mixture was stirred at reflux for 16 hours. The reactionmixture was cooled to room temperature, diluted with water (450 mL) andacidified to pH 1 with 1N HCl. Diethyl ether (200 mL) was added, and themixture was stirred for 15 minutes. The resultant precipitate wascollected by filtration and purified by flash silica gel chromatography(NH₄OH:MeOH:DCM, 0.5:5:94.5 to 2:20:78) to yield the ammonium salt as atan solid. The solid was suspended in water (100 mL) and, with rapidstirring, 1N HCl was added to pH 1. The mixture was stirred forapproximately 30 minutes, and then diethyl ether (50 mL) and ethylacetate (5 mL) were added and the mixture was stirred at roomtemperature for approximately 1 hour. The product was collected on afritted funnel (fine), washed with water (50 mL) and diethyl ether (50mL), and dried in vacuo at 40° C. overnight to provide 1.65 g (50%yield) of4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoicacid (1). ¹H NMR (DMSO-d₆) δ 12.08 (s, 1H), 10.23 (s, 1H), 8.72 (s, 1H),8.29 (d, 1H), 7.95 (br s, 1H), 7.80 (dd, 1H), 7.70 (d, 1H), 7.4-7.35 (m,2H), 7.21 (br s, 1H), 6.9 (br s, 2H), 4.9 (br s, 1H), 3.9 (br s, 1H),3.85 (s, 3H), 3.3 (br s, 3H); MS m/z 519 (M++H, 100%).

Compounds 2-18 were prepared by methods analogous to those described forcompound 1 or in Claiborne et al., WO 05/111039.

Example 2 Preparation of sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoatepolymorph form 1

To a stirred suspension of4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoicacid (98.0 g, 190 mmol) in ethanol (2.0 L) was added 1.044 M Sodiumhydroxide in water (199 mL). The resultant homogeneous solution wasstirred for 1 hour, during which time a thick precipitate formed. Theproduct was collected by filtration, and washed with ethanol (0.5 L) anddiethyl ether (1.0 L). The resultant solid was dried in vacuo at 60-70°C. for 4 days to provide 88.6 g (86.8%) of sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoateas a light tan solid, mp 225° C. (decomp). ¹H NMR (DMSO-d₆) δ 9.86 (s,1H), 8.60 (s, 1H), 8.29 (d, 1H), 7.79 (dd, 1H), 7.60 (br s, 1H), 7.40(dd, 1H), 7.29 (d, 1H), 7.25-7.15 (m, 2H), 6.9 (br s, 2H), 4.9 (br s,1H), 3.8 (br s, 1H), 3.70 (s, 3H), 3.35 (br s, 3H); MS m/z 519 (M⁺-Na+H,100%); CHN Anal. Calcd. for C₂₇H₁₉ClFN₄NaO₄.0.33 EtOH1.3 H₂O: C, 57.33;H, 4.10; N, 9.67. Found: C, 57.14; H, 3.99; N, 9.65.

Example 3 Preparation of sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoatepolymorph form 2

Sodium4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxybenzoatepolymorph form 1 (100 mg) was suspended in water (0.2 mL) and ethanol (2mL) and the mixture was stirred with heating at 70° C. for 6 hours. Themixture was cooled to room temperature, and the light yellow solid wascollected on a fritted funnel and dried in vacuo at 70° C. for 3 days toyield 70 mg of crystalline polymorph form 2, mp 265° C. ¹H NMR (DMSO-d₆)δ: 9.86 (s, 1H), 8.60 (s, 1H), 8.29 (d, 1H), 7.79 (dd, 1H), 7.60 (br s,1H), 7.40 (dd, 1H), 7.29 (d, 1H), 7.25-7.15 (m, 2H), 6.9 (br s, 2H), 4.9(br s, 1H), 3.8 (br s, 1H), 3.70 (s, 3H), 3.35 (br s, 3H). MS m/z 519(M+—Na+H, 100%).

Example 4 Expression and Purification of Protein Kinase Enzymes

Aurora A Enzyme Expression and Purification

Recombinant mouse Aurora A with an amino-terminus hexahistidine tag (SEQID NO: 1)(His-Aurora A) was expressed using a standard baculovirusvector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Soluble, recombinant mouse Aurora A was purified from insect cells usingNi-NTA agarose (Qiagen) as described by the manufacturer and furtherpurified over an S75 size exclusion column (Amersham Pharmacia Biotech).

Aurora B Enzyme Expression and Purification

Recombinant mouse Aurora B with an amino-terminus hexahistidine tag (SEQID NO: 1)(His-Aurora B) was expressed using a standard baculovirusvector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Soluble, recombinant mouse Aurora B was purified from insect cells usingNi-NTA agarose (Qiagen) as described by the manufacturer.

Example 5 Protein Kinase Enzyme Assays

Aurora A DELFIA® Kinase Assay

The mouse Aurora A enzymatic reaction totaled 25 μl and contained 25 mMTris-HCl (pH 8.5), 2.5 mM MgCl₂, 0.05% Surfact-AMPS-20, 5 mM SodiumFluoride, 5 mM DTT, 1 mM ATP, 3 μM peptide substrate(Biotin-β-Ala-QTRRKSTGGKAPR-NH₂)(SEQ ID NO: 2), and 0.5 nM recombinantmurine Aurora A enzyme. The enzymatic reaction mixture, with and withouttest compound, was incubated for 10 minutes at room temperature beforetermination with 100 μL of stop buffer (1% BSA, 0.05% Surfact-AMPS-20,and 100 mM EDTA). A total of 100 μL of the enzyme reaction mixture wastransferred to wells of a Neutravidin-coated 96-well plate (Pierce) andincubated at room temperature for 30 minutes. The wells were washed withwash buffer (25 mM Tris, 150 mM sodium chloride, and 0.1% Tween 20) andincubated for 1 hour with 100 μL of antibody reaction mixture containing1% BSA, 0.05% Surfact-AMPS-20, anti-phospho-PKA rabbit polyclonalantibody (1:2000, New England Biolabs), and europium labeled anti-rabbitIgG (1:2000, Perkin Elmer). The wells were washed and then the boundeuropium was liberated using 100 μL of Enhancement Solution (PerkinElmer). Quantification of europium was done using a Wallac™ EnVision(Perkin Elmer).

Aurora B DELFIA® Kinase Assay

The mouse Aurora B enzymatic reaction totaling 25 μL contained 25 mMTris-HCl (pH 8.5), 2.5 mM MgCl₂, 0.025% Surfact-AMPS-20 (Pierce), 1%Glycerol, 1 mM DTT, 1 mM ATP, 3 μM peptide substrate(Biotin-β-Ala-QTRRKSTGGKAPR-NH₂)(SEQ ID NO: 2), and 20 nM recombinantmurine Aurora B enzyme. The enzymatic reaction mixture, with or withouttest compound, was incubated for 3 hours at room temperature beforetermination with 100 μL of stop buffer (1% BSA, 0.05% Surfact-AMPS-20,and 100 mM EDTA). A total of 100 μL of the enzyme reaction mixture wastransferred to wells of a Neutravidin-coated 96-well plate (Pierce) andincubated at room temperature for 30 minutes. The wells were washed withwash buffer (25 mM Tris, 150 mM sodium chloride, and 0.1% Tween 20) andincubated for 1 hour with 100 μL of antibody reaction mix containing 1%BSA, 0.05% Surfact-AMPS-20, anti-phospho-PKA rabbit polyclonal antibody(1:2000, New England Biolabs), and europium labeled anti-rabbit IgG(1:2000, Perkin Elmer). The wells were washed and then the boundeuropium was liberated using 100 μL of Enhancement Solution (PerkinElmer). Quantification of europium was done using a Wallac™ EnVision(Perkin Elmer).

Example 6 Cellular Assay

pT288 Aurora A Autophosphorylation Assay.

Human tumor cells (HCT-116, obtained from ATCC) were grown on 96-welldishes in McCoy's 5A medium supplemented with 10% bovine calf serum and200 nM L-glutamine. After incubation, the growth medium was replacedwith 75 μL of fresh media and 25 μL of test compound was added to thecells in two-fold serial dilutions in dimethyl sulfoxide (DMSO) toachieve final concentrations ranging from 5 to 0.010 μM. Test compoundat each dilution was added as replicates in 4 rows on the dish, and DMSO(20 nM) was added to each well of two columns for the untreatedcontrols. The cells were treated with test compound or DMSO for 60minutes at 37° C. in a humidified cell culture chamber. Cells were thenfixed with 4% para-formaldehyde in phosphate-buffered saline (PBS) for10 minutes, permeated with 0.5% Triton X-100 in PBS for 10 minutes, andwashed twice in PBS.

Cells were stained with Phospho-Aurora 2/AIK (T288) rabbit antibody(1:60) and Anti-phospho-Ser/Thr-Pro, MPM2 mouse antibody (1:750)followed by Alexa 488-conjugated goat anti-rabbit IgG (1:180) and Alexa594-conjugated chicken anti-mouse IgG (1:180; Molecular Probes). Thecells were then stained with Alexa 488-conjugated chicken anti-goat IgG(1:180, Molecular Probes) and Hoechst (1:50,000). Cells were visualizedusing a Discovery-1 High Content Imaging System. Images from nine orsixteen sites per well were captured at 200× magnification. Inhibitionof Aurora A was determined by measuring pT288 (Aurora Aautophosphorylation) fluorescent intensity within MPM2 immunopositive(mitotic) cells using Metamorph software. Concentration response curveswere generated by calculating the decrease of pT288 fluorescentintensity in test compound-treated samples relative to the DMSO-treatedcontrols, and growth inhibition (IC₅₀) values were determined from thosecurves.

Compounds 1-28 all exhibited IC₅₀ values less than or equal to 0.03 μMin this assay. Compounds 1-8 exhibited IC₅₀ values less than or equal to0.01 μM in this assay.

BrdU Cell Proliferation Assay

Cell proliferation of each cell line was measured using the cellproliferation enzyme-linked immunosorbent assay (ELISA),5-bromo-2′-deoxyuridine (BrdU) colorimetric kit according to themanufacturer's recommendations. The assay measures cell proliferation byquantifying BrdU incorporation into replicating deoxyribonucleic acid(DNA). Briefly, each well was incubated with 10 μL of BrdU labelingreagent for 2 hours at 37° C. in a humidified cell culture chamber.After aspiration of the labeling media, the cells were fixed anddenatured by adding 200 μL of ethanol to each well and incubated for 30minutes at room temperature. The ethanol was aspirated and 100 μL ofperoxidase—conjugated anti-BrdU antibody (anti-BrdU-POD; 1:100 inantibody dilution buffer) was added to the cells. The cells wereincubated with the antibody for 90 minutes at room temperature. Thecells were then washed 3× with 250 μL of wash buffer/well and 100 μLtetramethyl-benzidine was added to each well. The cells were incubatedfor 15 to 30 minutes at room temperature prior to spectrophotometricanalysis.

A SpectraMax Plus 384 plate reader (Molecular Devices, Sunny ValeCalif.) was used to measure the absorbance of each well at 370 nm.Concentration response curves were generated by calculating the decreasein optical density in samples treated with test compound relative to theDMSO-treated controls.

Compounds 1-18 all exhibited LD50 values less than or equal to 0.1 μM inthis assay in HCT116 cells. Compounds 1-3, 5, 7-14, 17, and 18 allexhibited LD₅₀ values less than or equal to 1.0 μM in this assay inSW480 cells. Compounds 4and 6 were not tested.

Example 7 In Vivo Assays

In Vivo Tumor Efficacy Model

HCT-116 (1×10⁶) cells in McCoy's 5A medium were aseptically injectedinto the subcutaneous space in the right dorsal flank of female CD-1nude mice (age 8 weeks, Charles River) using a 23-ga needle. Tumorvolumes were calculated using standard procedures (0.5×(length×width²)).When the tumors reached a volume of approximately 200 mm³, mice weredosed orally with compound 1 or compound iii at various doses in avehicle of 10% HPbCD+1% NaHCO₃. Doses (0.1 mL) were administered via 22gauge oral gavage needle. Control animals received vehicle alone.Animals were dosed once daily for 21 days, and there were 10 animals ineach group. Tumor size and body weight were measured twice per week.Compounds 1 and iii were well-tolerated at all doses in this study. Ateach dose, compound 1 produced longer tumor growth delay [TGD=(time fortreated animals to reach average tumor volume of 1000 mm³)−(time forcontrol animals to reach average tumor volume of 1000 mm³)] and greatertumor growth inhibition [TGI=(average tumor volume of controlanimals−average tumor volume of treated animals)*100/(average tumorvolume of control animals)] than did compound iii.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, these particular embodiments areto be considered as illustrative and not restrictive. It will beappreciated by one skilled in the art from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention, which is to be defined by theappended claims rather than by the specific embodiments.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The issued patents, applications,and references that are cited herein are hereby incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. In the case ofinconsistencies, the present disclosure, including definitions, willcontrol.

What is claimed:
 1. A method for inhibiting Aurora kinase activity in acell, comprising contacting a cell with a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R^(a) isselected from the group consisting of C₁₋₃ aliphatic, C₁₋₃fluoroaliphatic, —R¹, —T—R¹, —R², and —T—R²; T is a C₁₋₃ alkylene chainoptionally substituted with fluoro; R¹ is an optionally substitutedaryl, heteroaryl, or heterocyclyl group; R² is selected from the groupconsisting of halo, —C≡C—R³, —CH═CH—R³, —N(R⁴)₂, and —OR⁵; R³ ishydrogen or an optionally substituted aliphatic, aryl, heteroaryl, orheterocyclyl group; each R⁴ independently is hydrogen or an optionallysubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or twoR⁴ on the same nitrogen atom, taken together with the nitrogen atom forman optionally substituted 5- to 6-membered heteroaryl or 4- to8-membered heterocyclyl ring having, in addition to the nitrogen atom,0-2 ring heteroatoms selected from N, 0, and S; R⁵ is hydrogen or anoptionally substituted aliphatic, aryl, heteroaryl, or heterocyclylgroup; and R^(b) is selected from the group consisting of fluoro,chloro, —CH₃, —CF₃, —OH, —OCH₃, —OCF₃, —OCH₂CH₃, and —OCH₂CF₃.
 2. Themethod of claim 1, wherein: R^(a) is selected from the group consistingof C₁₋₃ aliphatic, C₁₋₃ fluoroaliphatic, —R¹ and —R²; R¹ is a 5- or6-membered aryl or heteroaryl optionally substituted with onesubstituent selected from the group consisting of halo and C₁₋₃aliphatic; R² is selected from the group consisting of halo, —C≡C—R³,—CH═CH—R³, and —OR⁵; R³ is hydrogen, C₁₋₃ aliphatic or —CH₂OCH₃R⁵ ishydrogen or C₁₋₃ aliphatic; and R^(b) is selected from the groupconsisting of fluoro, —OH, —OCH₃, —OCF₃, and —OCH₂CF₃.
 3. The method ofclaim 1, wherein: R^(a) is halo, C₁₋₃ aliphatic, C₁₋₃ fluoroaliphatic,—OH, —O(C₁₋₃ aliphatic), —O(C₁₋₃ fluoroaliphatic), or —C≡C—R³,—CH═CH—R³; and R³ is hydrogen, C₁₋₃ aliphatic, or —CH₂—OCH₃; or R^(a) isa phenyl, furyl, pyrrolidinyl, or thienyl ring optionally substitutedwith one substituent selected from the group consisting of halo and C₁₋₃aliphatic.
 4. The method of claim 3, wherein: R^(a) is selected from thegroup consisting of chloro, fluoro, C₁₋₃ aliphatic, C₁₋₃fluoroaliphatic, —OCH₃, —C≡C—CH₃, —C≡C—CH₂OCH₃, —CH═CH₂, —CH═CHCH₃,N-methylpyrrolidinyl, thienyl, methylthienyl, furyl, methylfuryl,phenyl, fluorophenyl, and tolyl.
 5. The method of claim 1, wherein thecompound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.